RecipharmCobra Biologics, Keele (previously Cobra Biomanufacturing Plc) has been producing plasmid DNA for clinical trials for more than 10 years and has an approved site under the EU clinical trials directive. During this period, the company has produced more than 40 plasmids (ranging from 500 mg to 5 g) for 25 customers in Europe and the United States. These plasmids have been used for gene therapy and vaccines as well as to produce viral vectors. RecipharmCobra has developed its own manufacturing process and related technologies including an antibiotic-free plasmid maintenance system (ORT: operator repressor titration) and cell lysis technologies (1).

We assessed PlasmidSelect Xtra chromatography resin from GE Healthcare as part of an internal development program, the aim of which was to improve process robustness and resolution of key contaminants from the plasmid product. Although this work was part of a long-term development program, there was also an immediate requirement to address purity issues with on-going manufacturing projects in which the removal of some contaminants had proved problematic. Initial studies focused on introducing the GE resin as an additional polishing step to our existing platform process. Following the success of this work, studies were performed to identify whether it was possible to streamline the platform process to a two-step purification process that includes the PlasmidSelect Xtra resin.

PRODUCT FOCUS: GENE THERAPIES,

VACCINES, VIRAL VECTORS

PROCESS FOCUS: DOWNSTREAM PROCESSING

WHO SHOULD READ: PROCESS DEVELOPMENT AND MANUFACTURING, ANALYTICAL TESTING

KEYWORDS: CHROMATOGRAPHY, DNA

LEVEL: INTERMEDIATE

Scope of this development program was constrained by several process requirements. The process had to be:

• scalable and suitable for clinical production

• modifiable with minimal changes to existing recovery and capture steps

• economical (low cost of goods basis at manufacturing scale)

PlasmidSelect Xtra resin is based on thiophilic aromatic chromatography, which can separate plasmid from host contaminants (e.g., chromosomal DNA and endotoxin) and provides good resolution between open- and closed-circle forms of plasmid DNA (2,3). Separation was achieved using isocratic (as well as gradient) elution from the column, which is more easily scaled and may have better resolution and processing yields. The resin had reported binding capacities in the region of 2mg/mL, which is relatively high for a large macromolecule.

Background

Plasmid DNA is used in clinical trials two ways: as a therapeutic agent (either as a therapy or for the generation of vaccine antigens) and as a transfection agent to produce viral constructs such as lentivirus or adenoassociated virus (AAV), for which multiple plasmid constructs are required to produce an individual viral product in transient production systems. Plasmids used for therapeutic agents tend to be relatively small — usually 4.5–6 kb — but can be as small as 2.5 kb. Those used for viral constructs are larger (≥11 kb). Although the production of plasmids as therapeutic agents has always been performed to current good manufacturing practices (CGMPs), the regulations relating to their production for viral constructs has been under some debate. Within the Eurpean Union, however, the increasing trend seems to be that such plasmids should be produced under CGMPs from master cell banks and to the same standards and product-release specifications as are conventional plasmid products for therapeutic applications.

Production of plasmid DNA for clinical use creates purification challenges related to removal of bacterial host-cell contaminants, including chromosomal DNA, RNA, and endotoxins and the segregation of supercoiled and open-circle forms of the plasmid. Those contaminants have similar properties to the target molecule, so strategies must be developed for their removal. The FDA set recommendations for acceptable levels of contaminants for plasmid DNA at <1% w/w chromosomal DNA, RNA, and proteins, <40 EU endotoxin/mg plasmid, and >80% supercoiled pDNA (4). Those values are mainly guidelines; normal industry expectations for supercoiled plasmid levels is usually >90% and often as high as 95%.

Plasmid DNA is often regarded commercially as a commodity product, and many people assume that all plasmid contructs can be produced from a “platform process” with limited, if any process development. However, pDNA constructs are highly variable in plasmid size, plasmid backbone, and gene inserts, resulting in a wide range of cell culture productivity and highly variable levels of product and host-related contaminants that must be removed in the purification process. Therefore, a platform purification process applied to their production must be highly robust regarding key contaminant removal, and it should have an intrinsically low cost due to market expectations of product value.

The initial building blocks for a pDNA production process of high–copy-number plasmid fermentations and recovery of plasmid by alkali cell lysis are well established (5). Precipitation of contaminants from a lysate is used and may elminate the need to use bovine RNAseA for host RNA removal (6). A lysate is usually processed through several chromatographic steps for further contaminant removal. However, a nonchromatographic manufacturing process using selective precipitation with the detergent cetyltrimethyl-ammoniumbromide (CTAB) combined with silica absorption and ultraf iltration has also been described (7,9).

Chromatography separations are usually based on a combination of ion exchange (IEX), hydrophobic interaction (including ion-pair interaction), and size exclusion (SEC) chromatographies (9,10,11). These techniques can be manipulated to remove bulk contaminants such as RNA, host cell protein, and endotoxin. However, removal of host chromosomal DNA fragments and open-circle plasmids is more difficult to achieve because of their chemical similarities to the target supercoiled plasmid, and highly selective processes are required to achieve such separations at an industrial scale for a wide range of plasmid constructs.

The source and nature of those contaminants are poorly understood and complex to analyze. Generation of small host chromosomal DNA fragments that do not precipitate during alkali lysis is thought to be influenced by a number of factors, ranging from cell states in the fermentation to enzymatic and mechanical degradation during cell recovery and lysis. Furthermore, the level of host DNA will vary significantly by host strain and plasmid construct. There is also a limited understanding of why higher levels of open-circle plasmid forms are generated within some production systems. Although it is possible to manipulate fermentation conditions to reduce open-circle plasmids, levels will vary between plasmids. Larger plasmids are significantly more prone to the generation of high levels of open-circle plasmid than small ones.

Application of a platform approach to plasmid DNA production makes it infeasible to optimize individual operations to prevent the formation of these contaminants for each product produced. Consequently, the process requires high-resolution, robust steps that can consistently remove such contaminants from a wide range of feed streams.